In recent years, mouse and human iPS cells have been established one after another. Yamanaka et al. induced iPS cells by introducing the Oct3/4, Sox2, Klf4 and c-Myc genes into fibroblasts from a reporter mouse wherein the neomycin resistance gene is knocked-in into the Fbx15 locus, and forcing the cells to express the genes (1,2). Okita et al. (3) succeeded in establishing iPS cells (Nanog iPS cells) that show almost the same gene expression and epigenetic modification profiles as those in embryonic stem (ES) cells by producing a transgenic mouse wherein the green fluorescent protein (GFP) and puromycin-resistance genes are integrated into the locus of Nanog, whose expression is more localized in pluripotent cells than Fbx15 expression, forcing fibroblasts derived from the mouse to express the above-mentioned four genes, and selecting puromycin-resistant and GFP-positive cells. Similar results were obtained by other groups (4,5). Thereafter, it was revealed that iPS cells could also be produced with three factors other than the c-Myc gene (6).
Furthermore, Yamanaka et al. succeeded in establishing iPS cells by introducing the same four genes as those used in the mouse into human skin fibroblasts (1,7). On the other hand, a group of Thomson et al. produced human iPS cells using Nanog and Lin28 in place of Klf4 and c-Myc (8,9). Park et al. (10) produced human iPS cells using TERT, which is known as the human cell immortalizing gene, and the SV40 large T antigen, in addition to the four factors Oct3/4, Sox2, Klf4 and c-Myc. Hence, it has been demonstrated that iPS cells comparable to ES cells in terms of pluripotency can be produced in both humans and mice, by introducing defined factors into somatic cells.
However, the efficiency of iPS cell establishment is low at less than 1%. Especially, a problem of extremely low efficiency of iPS cell establishment arises when they are produced by introducing three factors (Oct3/4, Sox2, Klf4) other than c-Myc, which is feared to cause tumorigenesis in tissues and individuals differentiated from iPS cells, into somatic cells.
By the way, some reports are available on the association between the maintenance of the undifferentiated state and pluripotency of cells and hypoxic conditions. Ezashi et al. (11) observed that human ES (hES) cells cultured under hypoxic conditions had their differentiation suppressed, suggesting the necessity of cultivation under hypoxic conditions to maintain sufficient pluripotency for hES cells. Covello et al. (12) showed that a transcription regulatory factor induced early under hypoxic conditions (HIF-2α) was capable of inducing the expression of Oct3/4 and regulating the functions and differentiation of stem cells. Furthermore, Grayson et al. (13,14) showed that hypoxic conditions were involved in the maintenance of the undifferentiated state and pluripotency of human mesenchymal stem cells (hMSCs). However, no report is available on the relationship between the nuclear reprogramming process in somatic cells that have once differentiated and hypoxic conditions.